U.S. patent number 9,606,086 [Application Number 13/955,709] was granted by the patent office on 2017-03-28 for high-efficiency separation and manipulation of particles and cells in microfluidic device using surface acoustic waves at an oblique angle.
This patent grant is currently assigned to The Penn State Research Foundation. The grantee listed for this patent is The Penn State Research Foundation. Invention is credited to Xiaoyun Ding, Tony Jun Huang.
United States Patent |
9,606,086 |
Ding , et al. |
March 28, 2017 |
High-efficiency separation and manipulation of particles and cells
in microfluidic device using surface acoustic waves at an oblique
angle
Abstract
An apparatus for manipulating particles within a fluid sample
includes a substrate having a substrate surface. A surface acoustic
wave (SAW) generator generates a SAW within a SAW region of the
substrate surface. The SAW has an SAW direction aligned with a
pressure node. A channel is configured to receive the fluid sample
and the fluid sample has a flow direction which is at an oblique
angle to the SAW direction.
Inventors: |
Ding; Xiaoyun (State College,
PA), Huang; Tony Jun (State College, PA) |
Applicant: |
Name |
City |
State |
Country |
Type |
The Penn State Research Foundation |
University Park |
PA |
US |
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Assignee: |
The Penn State Research
Foundation (University Park, PA)
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Family
ID: |
50024161 |
Appl.
No.: |
13/955,709 |
Filed: |
July 31, 2013 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20140033808 A1 |
Feb 6, 2014 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61678214 |
Aug 1, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N
15/1459 (20130101); B01D 21/283 (20130101); G01N
29/222 (20130101); B01L 3/502761 (20130101); C12M
47/04 (20130101); G01N 29/02 (20130101); G01N
2015/1006 (20130101); B01L 2400/0436 (20130101); B01L
2200/0636 (20130101); B01L 2300/0864 (20130101); B01L
2200/0652 (20130101); G01N 2015/0019 (20130101); G01N
2291/02466 (20130101); G01N 2291/0423 (20130101) |
Current International
Class: |
G01N
29/02 (20060101); B01L 3/00 (20060101); G01N
15/14 (20060101); C12M 1/00 (20060101); G01N
29/22 (20060101); G01N 15/00 (20060101); G01N
15/10 (20060101) |
Field of
Search: |
;73/61.75,61.71,570.5,61.49,61.73,61.79 |
References Cited
[Referenced By]
U.S. Patent Documents
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2243630 |
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RU |
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2253888 |
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Jun 2005 |
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RU |
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WO-03089158 |
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Oct 2003 |
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WO |
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WO-2007128045 |
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Nov 2007 |
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WO |
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WO-2007128046 |
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Nov 2007 |
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WO |
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WO-2008083138 |
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Jul 2008 |
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WO |
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WO-2008118740 |
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Oct 2008 |
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WO |
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Other References
International Search Report and Written Opinon for corresponding
PCT Application No. PCT/US2013/052482 issued Nov. 21, 2013. cited
by applicant .
Nilsson, et al., Acoustic control of suspended particles in micro
fluidic chips, Lab on a Chip, 4:131-135, 2004. cited by applicant
.
Wang, et al., Single-molecule tracing on a fluidic microchip for
quantitative detection of low-abundance nucleic acids, Journal of
the American Chemical Society, 127:5354-5359, 2005. cited by
applicant .
Wang, et al., Dielectrophoresis switching with vertical sidewall
electrodes for microfluidic flow cytometry, Lab on a Chip,
7:1114-1120, 2007. cited by applicant .
Wiklund, et al., Ultrasonic standing wave manipulation technology
integrated into dielectrophoretic chip, Lab on a Chip, 6:1537-1544,
2006. cited by applicant .
Shi, et al., Focusing microparticles in a microfluidic channel with
standing surface acoustic waves (SSAW), Lab on a Chip, 8:221-223,
2008. cited by applicant .
Shi, et al., Acoustic tweezers: patterning cells and microparticles
using standing surface acoustic waves (SSAW), Lab on a Chip,
9:2890-2895, 2009. cited by applicant .
Mao, et al., Focusing fluids and light: enabling technologies for
single-particles detection in the micro/nanoscale, IEEE
Nanotechnology Magazine, 2:22-27, 2008. cited by applicant .
Mao, et al., "Microfluidic drifting"--implementing
three-dimensional hydrodynamic focusing with a single-layer planar
microfluidic device, Lab on a Chip, 7:1260-1262, 2007. cited by
applicant .
Mao, et al., Single-layer planar on-chip flow cytometer using
microfluidic drifting based three-dimensional (3D) hydrodynamic
focusing, Lab on a Chip, 9:1583-1589, 2009. cited by applicant
.
Wood, C.D. et al., "Alignment of particles in microfluidic systems
using standing surface acoustic waves," Applied Physics Letters,
2008, vol. 92, 044104 (Published online Jan. 30, 2008). cited by
applicant .
European Search Report, 52 pages, Jul. 4, 2016. cited by applicant
.
L. Johansson et al., "Surface Acoustic Wave Induced Particle
Manipulation in a PDMS Channel-Principle Concepts for Continuous
Flow Applications", Biomed Microdevices, (2012) 14:279-289. cited
by applicant.
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Primary Examiner: Fitzgerald; John
Assistant Examiner: Eyassu; Marrit
Attorney, Agent or Firm: McNees Wallace & Nurick LLC
Government Interests
STATEMENT OF GOVERNMENT SUPPORT
This invention was made with government support under Grant No.
OD007209, awarded by the National Institutes of Health, and under
Grant No. ECCS-0801922 awarded by the National Science Foundation.
The government has certain rights in the invention.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATION
This patent application claims priority from U.S. provisional
patent application Ser. No. 61/678,214, filed Aug. 1, 2012, the
content of which is incorporated herein in its entirety.
Claims
The invention claimed is:
1. An apparatus for sorting particles within a fluid sample, the
apparatus comprising: a substrate, having a substrate surface; a
surface acoustic wave (SAW) generator configured to generate a SAW
within a SAW region of the substrate surface, the SAW having a SAW
direction aligned with a generally linear pressure node; a channel,
configured to receive the fluid sample, the fluid sample having a
flow direction, the flow direction being at an oblique angle to the
SAW direction; and a plurality of output channels arranged and
disposed to collect a plurality of output particle streams from the
fluid sample in the channel.
2. The apparatus of claim 1, wherein: the SAW generator comprises a
pair of spaced apart surface acoustic wave generators, the surface
acoustic wave generators each being an interdigitated transducer
including interdigitated electrodes supported by the substrate.
3. The apparatus of claim 1, wherein: the substrate is a
piezoelectric substrate.
4. The apparatus of claim 1, wherein: the apparatus is a
microfluidic device; the channel being a microchannel having at
least one cross-sectional dimension less than 1 mm; and the
particles being microparticles having a cross-sectional dimension
less than 100 microns.
5. The apparatus of claim 1, wherein: the SAW generator is a
standing surface acoustic wave (SSAW) generator configured to
generate a SSAW within the SAW region.
6. An apparatus for sorting particles within a fluid sample, the
apparatus comprising: a substrate; a first surface acoustic wave
generator; a second surface acoustic wave generator, the first and
second surface acoustic wave generators being configured to
generate a surface acoustic wave (SAW) within a SAW region of the
substrate, the SAW having a SAW direction aligned with a generally
linear pressure node of the SAW; a channel configured to receive a
fluid sample including particles, the channel having a sorting
portion proximate the SAW region of the substrate, the channel
having a channel direction; and a plurality of output channels
arranged and disposed to collect a plurality of output particle
streams from the fluid sample in the channel, wherein the SAW
direction is disposed at an oblique angle to the channel direction,
and wherein by the apparatus is configured to sort particles within
the fluid sample into the plurality of output particle streams when
the fluid sample is introduced into the channel and the SAW is
generated.
7. The apparatus of claim 6, wherein: the SAW generator is a
standing surface acoustic wave (SSAW) generator configured to
generate a SSAW within the SAW region.
8. The apparatus of claim 6, wherein: the substrate is a
piezoelectric substrate; and the first and second surface acoustic
wave generators each comprise electrodes supported by the
substrate.
9. The apparatus of claim 6, wherein: the substrate forms a wall of
the channel.
10. The apparatus of claim 6, wherein: the apparatus is a
microfluidic device; the channel is a microchannel; and the
microchannel has at least one cross-sectional dimension less than 1
mm.
11. The apparatus of claim 6, wherein: the channel is a flow
channel configured to receive a sample fluid flow.
12. The apparatus of claim 6, wherein: the particles are
microparticles having a diameter of less than 100 microns.
13. The apparatus of claim 12, wherein: the microparticles include
biomolecules or cells.
14. A method of sorting a plurality of different types of particles
within a fluid sample including the plurality of different types of
particles, the method comprising: providing an apparatus for
sorting particles within a fluid sample, the apparatus comprising:
a substrate, having a substrate surface; a surface acoustic wave
(SAW) generator; a channel, configured to receive the fluid sample;
and a plurality of output channels; introducing the fluid sample to
the flow channel, the fluid sample including the plurality of
different types of particles and having a flow direction;
generating a SAW within a SAW region of the substrate surface, the
SAW having a SAW direction aligned with a generally linear pressure
node, the flow direction being at an oblique angle to the SAW
direction; sorting the plurality of different types of particles
into a plurality of output particle streams by type; and sorting
the plurality of output particle streams into the plurality of
output channels by type.
15. The method of claim 14, wherein: the generating a SAW step
comprises generating a standing surface acoustic wave (SSAW).
16. The method of claim 14, wherein: the fluid sample is a sample
flow directed along the flow channel; the flow channel is supported
by the substrate; the flow channel is a microchannel within a
microfluidic device; and the method further includes particle
characterization, particle focusing, particle separation, particle
fractionation, or particle selection.
17. The method of claim 14, wherein: the fluid sample includes a
first particle type being directed by pressure forces induced by
the SAW and a second particle type not being directed by the
pressure forces, so as to produce a first stream of the first
particle type and a second stream of the second particle type.
Description
FIELD OF THE INVENTION
The invention relates to methods and apparatus for particle or cell
manipulation, such as separation and focusing, and particle or cell
detection and characterization.
BACKGROUND OF THE INVENTION
Efficient separation of suspended particles and cells is essential
to many fundamental biomedical studies such as cancer cell
detection and drug screening. The most popular methods for cell
separation in the life science laboratory so far are the
centrifugal methods, which are capable of separating cells with
differences in size and density. Another industrial and clinical
standard for high quality cell separation is a FACS (fluorescence
activated cell sorter). The FACS technology is performed in a
sheath flow mode where cells are focused in the center of buffer
and then pass through a laser beam for high speed and precise
optical detection. The cells can be separated by a downstream
electric field triggered by the optical signal. In the past years,
fundamental advances in the lab-on-a-chip technologies have driven
development of new approaches to cell separation. Examples include
magnetic, hydrodynamic, optical lattice,
electrophoresis/dielectrophoretic (DEP), and acoustic methods.
The magnetic method starts with labeling cells of interest with
magnetic markers. Then an external magnetic field is applied to the
sample, leading to the separation of labeled cells from the rest.
The labeling step required for magnetic methods usually increases
cost and processing time, and may also have a negative effect on
the cells of interest. The hydrodynamic methods usually involve
high flow speed (inertial force based method) or asymmetric
obstacles inside the channel (deterministic lateral displacement).
These methods permit continuous operation without requiring
additional labeling or external forces. However, the channel
obstacles in the channel may exert high mechanical stress on cells
and lead to low throughput. The optical lattice method provides a
unique separation approach which can separate particles with
different optical properties. However, this approach has two
potential shortcomings: 1) the potential laser-induced heating, the
formation of singlet oxygen, and multiphoton absorption in
biological materials may cause physiological damage to cells and
other biological objects; and 2) the method relies on complex,
potentially expensive optical setups that are difficult to maintain
and miniaturize. Electrophoresis/dielectrophoresis based methods
are strictly dependent on particle polarizibility and medium
conductivity, and utilize electrical forces that may adversely
affect cell physiology due to current-induced heating and/or direct
electric-field interaction.
Acoustic-based particle manipulation methods present excellent
alternatives. Compared to their optical, electrical, or magnetic
counterparts, acoustic-based methods are relatively non-invasive to
biological objects and work for most microparticles regardless of
their optical, electrical, or magnetic properties. The well
developed bulk acoustic wave (BAW) acoustophoresis has demonstrated
the separation of cells based on size and density in microfluidic
chips without any labeling on the target particles or cells. This
BAW method, however, requires a channel material with excellent
acoustic reflection properties (such as silicon and glass). The
widely used soft polymer materials in microfluidic applications,
such as PDMS, usually do not have those properties. Moreover, the
transducer to generate BAW is bulky and hinders the system
integration and miniaturization.
SUMMARY OF THE INVENTION
The present invention provides a unique design based on a surface
acoustic wave method. Some versions demonstrate a high separation
efficiency with separation efficiency of 98% or higher. Cell
viability, proliferation, and apoptosis tests were carried out to
confirm the excellent biocompatibility of this device.
An example apparatus for separating particles within a fluid sample
comprises a substrate, one or more transducers for generating a
surface acoustic wave (SAW) in the substrate, and a channel
configured to receive a fluid sample including one or more species
of particle. The fluid sample may be a sample fluid flow, and the
sample fluid flow may have a focused, separated, or otherwise
sorted particle stream after passing through the particle
manipulation portion of the channel. The channel direction or flow
direction is at an oblique angle to the direction of the SAWs. The
SAWs may be standing surface acoustic waves (SSAWs)
Examples of the present invention provide novel methods and
apparatus for high-efficiency separation of micro/nano particles
and cells using angled or tilted surface acoustic waves on a
single-layer planar microfluidic device fabricated using standard
soft-lithography techniques. Systems include a low cost, high
efficiency, and portable separation system for many applications
such as blood/cell/particle separation, cells/particles medium
exchange, and cells/particles enrichment.
A channel has a particle manipulation portion where the channel is
proximate a SAW region of the substrate, for example extending over
the SAW region. The SAW region can be defined using a patterned
material on the substrate. The channel may be provided by a formed
element, such as a molded polymer formed element, adjacent the
substrate. The particle manipulation portion of the channel
provides particle manipulation within the fluid sample when a
surface acoustic wave is generated. The fluid sample may comprise
particles suspended in a liquid, such as an aqueous medium.
In some examples, the substrate is a piezoelectric substrate, and
the SAW is generated using a transducer supported by the substrate.
A standing surface acoustic wave (SSAW) may be generated using a
pair of surface acoustic wave generators (SAW generators), which
may each be an interdigitated transducer (IDT). The SAW generators
may be spaced apart on the substrate, and the SAW region of the
substrate is located where SAWs interact on the surface. In some
examples, a pair of SAW generators is used, and the particle
manipulation region of the channel is located between the SAW
generators, e.g. mechanically coupled to a SAW region of the
substrate so that the SAW generates pressure forces within the
fluid sample.
Example apparatus include microfluidic devices, the channel being a
microchannel having at least one cross-sectional dimension (such as
width or height) less than 10 mm, or less than 1 mm for some
versions, for example between 1 micron and 500 microns, and the
particles may be microparticles such as cells, biomolecules,
polymer beads, blood components such as red and white blood cells,
platelets, proteins, and the like.
An apparatus may be a particle characterization apparatus further
including a particle characterization device, the particle
characterization device characterizing the manipulated particles.
Particle characterization may include counting, sorting, detecting
(including selective detection of one or more particle species), or
otherwise characterizing particles, and may include diagnosis of a
human disorder based on the presence or properties of a biological
fluid component. Examples include blood, saliva, urine, and other
biological fluid characterization including manipulation of
particles within the biological fluid. A particle characterization
apparatus may include a radiation source providing a radiation beam
incident on the manipulated particles, and/or a sensor receiving
radiation scattered or otherwise obtained from the particles.
Example particle characterization apparatus include a cytometer
(such as a flow cytometer), fluorescence particle detector,
fluorescence spectrometer, fluorescence-activated particle sorter,
other particle sorter, particle counter, fluorescent spectrometer,
biomarker detector, or genetic analyzer. Particles may be cells
(e.g. human cells), biomolecules, other bioparticles, or any other
type of particle of interest.
An example method of particle manipulation within a fluid sample
including the particles comprises introducing the fluid sample to a
channel proximate a substrate, and generating a SAW or SSAW on the
substrate at an oblique angle to the channel direction. A SAW is an
acoustic wave propagating along the surface of the substrate, and
the surface may also be in contact with a fluid sample. The SAWs
may interact to form a SSAW. The term acoustic does not limit the
frequency of the SAW, which may greater than 1 GHz. Manipulated
particles may be particles within a region of enhanced particle
concentration within a liquid.
The SAW induces pressure forces within the fluid so as to focus the
particles within the fluid sample. The sample flow may be directed
along a flow channel, the flow channel being supported by the
substrate in which the SAW is generated. A SAW may be used to
obtain three-dimensional manipulation of the particles within the
sample flow, the particles being manipulated in directions both
parallel and normal to the substrate.
A novel on-chip micro/nano particle manipulation technique was
developed using standing surface acoustic waves (SSAWs). Example
methods and apparatus are efficient, simple, fast, dilution-free,
and applicable to virtually any type of particle, including both
charged and uncharged microparticles. Example methods can be used
with flow cytometry, cell sorting/counting, on-chip cell
manipulation, tissue engineering, regenerative medicine, non-human
animal diagnosis, and many other applications.
An example apparatus, such as a microfluidic device, receives a
sample flow including particles. The apparatus comprises a
substrate, a channel (such as a flow channel) into which the sample
is introduced, and one or more surface acoustic wave (SAW)
generators. A SAW generator may be an interdigitated transducer
(IDT, sometimes termed an interdigital transducer) comprising
interdigitated comb-shaped electrodes on a piezoelectric substrate.
The channel may pass between a pair of IDTs. The IDTs and channel
may both be supported by the same piezoelectric substrate. The SAW
generators may be operated to produce a SAW or SSAW in a portion of
the substrate proximate (possibly immediately adjacent to) the
manipulation portion of the flow channel. For example, a flow
channel may be supported by the substrate, e.g. formed by a
structure comprising a polymer or other material bonded to the
substrate.
The flow channel has a particle manipulation region located on a
portion of the substrate in which the SAW exists. For example, the
flow channel may pass over a portion of the substrate having
standing surface acoustic waves (SSAWs), the particles being
manipulated within the flow channel by the effects of the SSAW. The
substrate may be a generally planar substrate, for example a
ferroelectric and/or piezoelectric substrate. A surface acoustic
wave generator may comprise interdigitated electrodes supported by
a ferroelectric or piezoelectric substrate. Two or more SAW
generators may be used to generate a SSAW in the substrate, e.g.
using interference effects between SAWs.
A method of manipulating particles within a sample, such as
focusing, separating, or sorting, which may be a method of
three-dimensional particle manipulation, includes producing a
standing surface acoustic wave (SSAW), pressure waves within the
sample generated as a result of the SSAW producing particle
manipulation. The sample may be a sample flow moving through a
channel, the channel having a particle manipulation region over a
portion of the substrate in which the SSAW exists.
An apparatus for three-dimensional particle manipulation of
particles within a fluid sample comprises a substrate having a
substrate surface, a surface acoustic wave generator, operable to
generate a surface acoustic wave (SAW, such as a SSAW) within a SAW
region of the substrate surface, a channel configured to receive
the fluid sample, the channel having a particle manipulation
portion proximate the SAW region of the substrate, the particle
manipulation portion providing manipulated particles within the
fluid sample when the SAW is generated. The substrate surface may
form a wall of the channel, and the SAW region of the substrate may
form a wall of the particle manipulation portion of the
channel.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic top view of a particle manipulation apparatus
in accordance with the present invention;
FIG. 2 is a cross sectional schematic view showing the interaction
of SSAWs with particles in a channel;
FIGS. 3A-3C illustrate the trajectories taken by two different
particle types in the presence of SSAWs disposed at an oblique
angle of 15, 30 and 45 degrees, respectively, to the direction of
flow;
FIGS. 4A-4C illustrate the trajectories taken by a particle in the
presence of SSAWs having three different input power levels;
FIG. 5 is a schematic view similar to FIG. 1, with a working region
indicated in dot-dash lines;
FIG. 5A illustrates the trajectories of two particle types in a
fluid flow in the working region of FIG. 5, with the SAW generators
turned off;
FIG. 5B illustrates the trajectories of two particle types in a
fluid flow in the working region of FIG. 5, with the SAW generators
turned on;
FIG. 5C is a schematic view similar to FIG. 5A, with an outlet
region indicated in dot-dash lines;
FIG. 5D illustrates the trajectories of two particle types in a
fluid flow in the outlet region of FIG. 5C, with the SAW generators
turned off;
FIG. 5E illustrates the trajectories of two particle types in a
fluid flow in the outlet region of FIG. 5C, with the SAW generators
turned on;
FIG. 6 is a graph presenting experimental data on the separation
efficiency of the present invention;
FIG. 7 is a schematic view similar to FIG. 1, with a working region
indicated in dot-dash lines;
FIG. 7A illustrates the trajectories of two particle types in a
fluid flow in the working region of FIG. 7, in the presence of
SSAWs;
FIG. 7B is a schematic view similar to FIG. 7, with an outlet
region indicated in dot-dash lines;
FIG. 7C illustrates the trajectories of two particle types in a
fluid flow in the outlet region of FIG. 7B, in the presence of
SSAWs;
FIG. 8 is a schematic view similar to FIG. 1, with a working region
indicated in dot-dash lines;
FIG. 8A illustrates the trajectories of two particle types in a
fluid flow in the working region of FIG. 8, in the presence of
SSAWs;
FIG. 8B is a schematic view similar to FIG. 8, with an outlet
region indicated in dot-dash lines;
FIG. 8C illustrates the trajectories of two particle types in a
fluid flow in the outlet region of FIG. 8B, in the presence of
SSAWs;
DETAILED DESCRIPTION OF THE INVENTION
Example apparatus and methods using a novel acoustic manipulation
technique using surface acoustic waves (SAW), in particular
standing surface acoustic waves (SSAW), allow fast and effective
particle manipulation. Examples include apparatus and methods for
microparticle manipulation inside a microfluidic channel. Example
approaches are simple, fast, dilution-free, and can be used to
focus virtually any microparticles, including both charged and
uncharged particles. The transparency of the particle manipulation
device makes it compatible with most optical characterization tools
used in biology and medicine, allowing particle characterization by
fluorescence and/or other optical techniques. A surface acoustic
wave (such as a SSAW) can be used for manipulation of arbitrary
particles, such as micro/nano particles, and for example particle
focusing within a fluid flow, particle sorting or separation.
Throughout this disclosure, reference is made to surface acoustic
waves (SAWs). It should be understood that standing surface
acoustic waves (SSAWs) are one type of SAW, and are preferred for
some embodiments. However, the present invention is not limited to
SSAWs, as other types of SAWs may be used for some versions. Some
versions will be described as utilizing SSAWs, but further versions
may use other types of SAWs. An example of another type of SAW is a
traveling surface acoustic wave (TSAW).
A SAW-based technique localizes most of the acoustic energy on the
surface of the substrate, and has little loss along the propagation
line, lowering the power consumption and improving the uniformity
of the standing waves. The SAW technique is compatible with
standard soft lithography techniques, and can be used in a wide
variety of on-chip biological/biochemical applications. In
experimental examples, a standing surface acoustic wave (SSAW)
manipulation technique was used with a microfluidic device using a
PDMS channel fabricated by standard soft lithography, and the SSAW
was directed at an oblique or tilted direction to the flow channel
elongation direction and the flow channel.
Examples of the present invention provide a novel method to
implement the high-efficiency separation of micro/nano particles
and cells using angled or tilted surface acoustic waves on a
single-layer planar microfluidic device fabricated using standard
soft-lithography technique. Compared to currently existing
technologies (e.g. bulk acoustic wave based separation, magnetic
field based separation and electrokinetic separation), this
technique provides a higher efficiency, significant simplification
for device fabrication, less invasiveness, and reduction of cost.
Systems include in a low cost, high efficiency, and portable
separation system for many applications such as blood component
separation, cell separation, particle separation, cells/particles
medium exchange, cell enrichment, and other particle enrichment.
Certain species or characteristics of particles, such as cells, may
be physically separated from a common stream to give a plurality of
exit streams, sorted by species or some characteristic. As used in
examples herein, a particle may be a biological cell unless
otherwise defined, but the term cell is sometimes used separately
to emphasize biological applications.
To date, many methods capable of particle and cell separation in
microfluidic systems, such as centrifugal methods, magnetic force,
hydrodynamic force, dielectrophoretic (DEP), and bulk acoustic
waves (BAW) have been developed. Particle separation is possible
through standing surface acoustic wave (SSAW)-induced
acoustophoresis in a microfluidic channel, with a separation
efficiency of 85% achieved. An angled or tilted interdigital
transducer (TIDT) based particle separation technique in accordance
with the present invention has demonstrated a remarkable separation
efficiency of 98% or higher.
An exemplary apparatus for manipulating particles is shown at 10 in
FIG. 1. A channel 12 is defined between a pair of spaced apart
surface acoustic wave generators 14 and 16. The generators 14 and
16 together define a SSAW generator. In the illustrated example,
the generators 14 and 16 are interdigitated transducers (IDTs). The
surface acoustic waves from the generators 14 and 16 interact to
form SSAWs therebetween. In FIG. 1, the SSAWS are indicated
generally at 18, and have nodes indicated by solid lines at 20, 22
and 24 and anti-nodes indicated by dashed lines at 26 and 28. The
central node is at 22. An SAW region may be considered to be the
area where SSAWs are generated between the generators 14 and 16.
The channel passes through the SAW region.
FIG. 2 is a cross sectional schematic view, taken along the central
node 22. The channel 12 is defined inside a channel wall 30. The
term "channel" may refer to the passage or the enclosing structure
in this description. Particles 32 are shown being urged toward the
antinode 22.
A fluid containing particles flows along the channel 12 in a flow
direction indicated at F. This direction may also be considered a
channel direction. The SSAWs may be said to have a SSAW direction,
which lies along the line 22 in FIG. 1. That is, the SSAW direction
is a direction aligned with the generally linear anti-nodes and
nodes of the SSAWs. As shown, the SSAW direction is at an oblique
angle to the flow direction or channel direction F. The SSAW
direction is neither parallel to nor perpendicular to the flow and
channel direction F. As known to those of skill in the art, oblique
defines an angle between 0 and 90 degrees and between 90 and 180
degrees, not including 0, 90 or 180. As will be clear to those of
skill in the art, SAWs other than SSAWs will also have nodes and
anti-nodes, though they may be positioned differently than shown
and may move over time. However, they will still occur at the angle
indicated, with respect to the flow or channel direction F.
An example of a SSAW based separation device consists of a
polydimethylsiloxane (PDMS) microfluidic channel 30 bonded in
between an identical pair of IDTs deposited on a piezoelectric
substrate 34. In FIG. 1, the microfluidic channel has three inlets
36, 38, and 40, and two outlets 42 and 44. The center inlet 38
introduces a fluid sample, containing particles to be manipulated,
and the two side inlets introduce a buffer flow. In experimental
arrangements, the pair of IDTs 14 and 16 were deposited in a
parallel arrangement, and aligned at a specific oblique angle, a.
Angles of 15.degree., 30.degree., and 45.degree. to the channel and
flow direction were used, but other oblique angles are possible. A
RF signal was applied to each IDT to generate two identical SAWs.
These two SAWs propagate in opposite directions and interfere with
each other to form a standing SAW (SSAW) in between the IDTs where
the PDMS microchannel 30 was bonded. Such a SSAW generates a
parallel distribution of pressure nodes and antinodes on the
surface of the substrate 34. The acoustic radiation force,
generated from the pressure distribution, pushes the suspended
particles towards pressure nodes or antinodes in the SSAW field,
depending on the elastic properties of the microparticles. FIG. 2
shows how the particles are pushed towards the pressure node.
Particles are injected through the center inlet channel 38 and
hydrodynamically focused by two side flows from side inlets 36 and
40 before entering the SSAW field. Particles in this SSAW field
experience lateral acoustic radiation force, drag force, gravity
force and buoyant force. Gravity force and buoyant force are
similar in magnitude but opposite in direction, and are almost
balanced. The behavior of particles in the channel can be
characterized by examining the drag force and acoustic radiation
force.
The primary acoustic radiation force (Fr) and drag force (Fd) can
be expressed as
.times..times..times..times..beta..times..times..lamda..times..PHI..funct-
ion..beta..rho..times..function..times..times..PHI..function..beta..rho..t-
imes..times..rho..times..times..times..times..rho..rho..beta..beta..times.-
.times..pi..times..times..eta..times..times. ##EQU00001## where
.rho..sub.0, .lamda., V.sub.p, .rho..sub.p, .rho..sub.m,
.beta..sub.p, .beta..sub.m, .eta., r, and v are the acoustic
pressure, wavelength, volume of the particle, density of the
particle, density of the medium, compressibility of the particle,
compressibility of the medium, medium viscosity, particle radius,
and relative velocity, respectively. Equation (2) describes the
acoustic contrast factor, .phi., which determines whether the
particles move to pressure nodes or antinodes: the particles will
aggregate at pressure nodes when .phi. is positive and pressure
antinodes when .phi. is negative. It is believed that most
particles and cells have positive .phi., and go to pressure nodes
in the SSAW fields, bubbles and lipids usually have negative .phi.
and move to pressure anti-nodes. Equations (1) and (3) indicate
that the radiation acoustic force is proportional to the volume of
the particle/cell while the drag force is proportional to the
radius of particle. Large particles that experience larger acoustic
force will be confined in the pressure node, and will be
repositioned with large lateral displacements along the width of
the channel. FIG. 1 shows larger particles being repositioned so as
to alight with the pressure node 22. These larger particles are
collected in upper outlet channel 42. For the small particles, the
forces acting on them are not large enough to confine them in the
pressure node. Therefore, they remain in the center stream by the
drag force and are collected in the bottom outlet channel 44, as
shown in FIG. 2.
FIGS. 3A-3C illustrate the trajectories taken by 15 .mu.m and 7
.mu.m polystyrene particles in the presence of SSAWs disposed at an
oblique angle of 15, 30 and 45 degrees, respectively, to the
direction of flow. The solid-looking line at 46 represents the
larger particles while 48 indicates the flow of smaller particles
in each Figure.
At a high input power, corresponding to large SSAW amplitude,
acoustic radiation forces dominate and confine the particle
trajectory along the angled pressure node, such as 22 in FIG. 1,
achieving a large distance shift across the width of the channel.
Low input power leads to small acoustic radiation forces and drag
force dominates on the particles, causing a small lateral distance
shift. The trajectory of 15 .mu.m particles at different SSAW
amplitude was experimentally recorded at a flow velocity of
.about.2 mm/s, as shown in FIGS. 4A-4C. FIG. 4A represents an input
power of 27 dBm, FIG. 4B represents an input power of 23 dBm, and
FIG. 4C represents an input power of 15 dBm. Since the acoustic
radiation force depends on the mechanical properties such as
volume, compressibility, and density, particles with differences in
those properties can be differentiated and separated by the
acoustic device described herein.
An embodiment of the present invention was tested using polystyrene
beads. Positions in the SAW working region and outlet of the
channel were recorded to analyze the distribution of the particles,
as shown in FIGS. 5-5E. FIG. 5 shows the device 50 with a working
region indicated in dot-dashed lines at 52. A mixture of 10 .mu.m
and 2 .mu.m particles were injected into the channel and were
hydrodynamically focused in the center of the channel by two side
flows. FIG. 5C shows the device and an outlet region at 54. When
generators were turned off, small particles and big particles were
flowing together along the stream and exited through the lower
outlet channel, as shown in FIGS. 5A and 5D. When the generators
were turned on, particles entering the working region 52
experienced acoustic radiation force, which pushed them towards the
linear pressure nodes tilted with an angle of 30.degree. with
respect to the flow direction. At a flow velocity of 6.5 mm/s and
input power of 16-23 dBm, the acoustic radiation forces pushed
large particles towards the pressure node and confined them along
the angled linear node until those particles exited the working
region, as shown in FIG. 5B. The small particles, however, due to
the insufficient acoustic radiation force acting on them, remained
in the original flow stream. FIGS. 5B and 5E indicate that large
particles were pulled out from the mixture stream and were
separated through the upper outlet channel while small particles
trajectory were not significantly affected and were collected in
the lower outlet channel. The ratio of large and small particles
collected from each outlet channel was analyzed to evaluate this
method. The number of particles was counted through a recorded
video. 98% of the large particles migrated to the upper outlet
channel while 100% of the small particles remained in the lower
outlet channel, as shown in FIG. 6.
To further examine the resolution of the technique, fluorescent
polystyrene beads with diameters of 9.9 .mu.m and 7.3 .mu.m were
mixed into an aqueous buffer. A mixture of those beads were
injected into the device and set to flow at .about.1.5 mm/s. The
small beads and large beads were mixed before entering the SSAW
working region. The large beads were extracted from the small beads
stream while passing through the working region. The fluorescent
intensity profile was scanned near the outlet channel to indicate
the beads distribution. The results showed two peaks for small
beads, which was caused by the non-uniform flow velocity
distribution in the vertical direction. This is attributable to the
hydrodynamic effect within the laminar flow. The experimental
result shows that this method achieved the separation resolution of
30%, which is better than most of other methods.
To further explore the versatility of the inventive method,
particle separation was carried out based on the difference of
compressibility. HL-60 are a human promyelocytic leukemia cell
line, with a diameter of .about.15 .mu.m. H1-60 cells (with a
density of .about.1.075 kg m.sup.-3, compressibility of
.about.4*10.sup.-10 Pa.sup.-1) were mixed with 15 .mu.m polystyrene
beads (with a density of 1.05 kg m.sup.-3, compressibility of
.about.2.16*10.sup.-10 Pa.sup.-1). These particles have similar
sizes and densities but different compressibilities. FIGS. 7-7C
show the separation of particles with different compressibilities.
FIGS. 7 and 7B show an exemplary device 60 with a working region at
62 and an outlet region at 64. FIGS. 7A and 7C shown the separation
in the working region and outlet region, respectively. Polystyrene
beads (dark circles) were pulled out of HL-60 cells (dotted
circles) in the SSAW working region and eventually collected by
upper outlet channel.
To demonstrate the ability of the inventive device for biological
applications, an experimental separation of human leukemia cancer
cells from human blood was carried out. Human red blood cells
(purchased from Zen-bio) were diluted with PBS (Phosphate buffered
saline) buffer by 100 times and mixed with HL-60 (human
promyelocytic leukemia cells). The ratio of blood cells and HL-60
was close to 1 to 1. FIGS. 8-8C represent stacked images showing
the cell separation process, in which HL-60 cells were selectively
moved from the red blood cells and collected from the upper outlet
channel. FIGS. 8 and 8B show an exemplary device 70 with a working
region shown at 72 and an outlet region shown at 74. FIGS. 8A and
8C shown the separation in the working region and the outlet
region, respectively. To evaluate the separation efficiency, cells
were collected by each outlet channel and then characterized using
commercial standard flow cytometry (Coulter FC 500). As a
comparison, the mixture sample was also counted through the flow
cytometry. The results show that 82% of the cells from upper outlet
channel were HL-60 and 81% were red blood cells from the lower
outlet channel.
Circulating tumor cells (CTCs) have drawn increasing research
attention in recent years due to their potential value in cancer
prognosis, therapy monitoring, and metastasis research. Rare CTCs
in the blood of patients with metastatic cancer are a potentially
accessible source for detection, characterization, and monitoring
of non-hematological cancers. The isolation of CTCs is a tremendous
technical challenge due to their low concentration, as few as one
cell per 10.sup.9 haematological cells in blood.
To demonstrate the applicability of the present invention to CTC,
the inventors studied isolation of cancer cells from human blood.
In the study, 1 mL human whole blood was lysed using RBC Lysis
Buffer [eBioscience], and the white blood cells (WBC) concentration
was measured to be 2-4*106/mL. This erythrocyte-lysed blood sample
was then mixed with 100 uL cancer cell (6*106/mL) to achieve a
cancer cell concentration of 10%. Here MCF-7 cells (human breast
cancer cell line) were used as a cancer cell model. The mixed
sample was then delivered into a SSAW-based CTC isolation device
through a syringe pump. Since cancer cells are usually much larger
than white blood cells, when the cells entered the SSAW working
region, cancer cells were isolated from WBCs. CTC cells and
leukocytes are eventually collected from different outlets for
consecutive characterization. EpCAM, CD45 surface markers (green),
and a nuclear stain (DAPI, blue) were used to investigate the
purity of isolated CTC. Epithelia cancer cells such as MCF-7 are
positive to EpCAM (red), negative to CD45, and positive to DAPI
(blue), while leukocytes are negative to EpCAM, positive to CD45,
and positive to DAPI (blue). To evaluate the performance of cancer
cell isolation using the inventive device, the recovery rate and
purity of cancer cell isolation were investigated. The recovery
rate (%) and purity (%) of cell isolation are defined as the
percentage of the isolated cancer cell number over the spiked
cancer cell number and that of the isolated cancer cell number over
the total collected cell number, respectively. The MCF-7 cell line
was used as the CTC model, and the preliminary result indicated a
purity as high as 98%, much higher than that of the current
commercial approach, Cellsearch (0.1%), and higher than that of
other state-of-art label free CTC isolation methods (80%-90%).
Biocompatibility of the inventive CTC isolation device is very
important since further CTC cell physiological studies will be
conducted after CTCs are collected. Therefore, it is required for
the isolation process to have very little, if any, physiological
impact on the cells. To demonstrate the biocompatibility of the
inventive device, cells viability, apoptosis, and proliferation
assays were performed after exposure to an SAW field at a working
power level (25 dbm, or 2 W/cm2). The WST-1 cell viability test
(Roche), BrdU Cell Proliferation ELISA (Roche), and Calcein AM and
SYTOX Orange (Invitrogen) were used to test cells viability,
proliferation, and apoptosis, respectively. MCF-7 cells were
delivered into the separation device at a flow rate of 2 uL/min
under the input power of 25 dBm (2 W/cm2). Cell tests were then
conducted immediately after being collected from the outlet. The
results indicate that no significant changes were found in cell
viability, apoptosis and proliferation. These promising results
show that the inventive SAW device is ideal for CTCs isolation from
blood for consecutive CTCs study without affecting cell
physiological properties.
Fresh human whole blood with Acid Citrate Dextrose (ACD) as
anticoagulant was purchased from Zen-bio. To lyse the red blood
cells, 1 ml of whole blood was incubated with 10 ml of 1.times.RBC
Lysis Buffer (eBioscience) for 10-15 min at room temperature
followed by centrifugation at 400.times.g, resuspension in PBS, and
cell counting with Hemacytometer to determine white blood cell
(WBC) concentration. Then cultured MCF7 breast cancer cells were
spiked into the prepared WBC suspension at a desired ratio. This
prepared sample was injected into the inventive SSAW device for
MCF7 separation.
After separation, cells from the CTC outlet were collected and
fixed with 4% paraformaldehyde (Santa Cruz Biotechnology, Inc.) for
5 min and subsequently permeabilized with 0.2% Triton X-100
(Sigma-Aldrich) in PBS. These fixed cells were then stained with
DAPI (nuclei staining), FITC-conjugated anti-CD45 antibody (WBC
staining) (Invitrogen), and Phycoerythrin (PE)-conjugated
anti-EpCAM antibody (MCF7 staining) (eBioscience). The stained
cells were analyzed through epifluorescence imaging.
The present invention provides a unique cell separation
microfluidic device using standing surface acoustic wave. Particles
of varying size and compressibility can be effectively and
continuously separated using this device. The inventors have
successfully demonstrated on-chip continuous separation of 1)
polystyrene beads with different size, 2) beads and cells with same
size but different compressibility, 3) Leukemia cancer cells from
human red blood cells, and 4) Human breast cancer cells from Human
white blood cells as CTCs model. A series of cells viability,
proliferation, and apoptosis tests were performed to prove
excellent biocompatibility of the inventive method. In addition,
the inventive SSAW device is simple, low cost, miniaturized, and
can be fabricated via standard microfabrication, allowing the easy
integration into other lab-on-chip technologies.
Examples of the invention provide novel apparatus and methods to
implement the high-efficiency separation of micro/nano particles
and cells using oblique angled standing surface acoustic waves on a
single-layer planar microfluidic device fabricated using standard
soft-lithography technique. Compared to currently existing
technologies (e.g. bulk acoustic wave based separation, magnetic
field based separation and electrokinetic separation), this
technique provides a higher efficiency, significant simplification
for device fabrication, less invasiveness, and reduction of cost.
Examples of the novel system include low cost, high efficiency, and
portable separation system for many applications such as
blood/cell/particle separation, cells/particles medium exchange,
and cells/particles enrichment.
An example apparatus for manipulating (sorting, separating,
focusing, or otherwise manipulating) particles within a fluid
sample includes a substrate, having a substrate surface; and an
acoustic transducer such as an IDT, operable to generate an e.g.
standing surface acoustic wave (SSAW) within a region of the
substrate surface. A channel is configured to receive a fluid
sample. For example, the channel may be a flow channel configured
to receive a fluid sample having a flow direction. The flow
direction may be at an oblique angle to the SSAW direction, e.g. at
least 5 degrees from parallel or perpendicular to the flow
direction, for example at least 10 degrees from parallel or
perpendicular to the flow direction. For example, the angle between
the SSAW and the channel direction may be between 5 and 85, such as
between 10.degree. and 80.degree., for example between 10.degree.
and 70.degree.. These angular ranges are exemplary and not
limiting. The SSAW generator may include a pair of spaced apart
surface acoustic wave generators, and the surface acoustic wave
generators may each be an interdigitated transducer (IDT) including
interdigitated electrodes supported by the substrate. The substrate
may be, or include, a piezoelectric substrate. The SAWs generated
by the pair of transducers may be parallel to each other, with
opposed direction, to form a SSAW extending between the transducers
and at an oblique direction to the flow channel. A flow channel
passes between the transducers and proximate an SSAW formed between
them. Differing pressure forces on different particle types may be
used to form a plurality of output particle streams downstream from
the SSAW region. These output particle streams may then be
collected by a plurality of output channels, each output channels
collecting a stream of particular particle type.
An example apparatus may be a microfluidic device, the channel
being a microchannel having at least one cross-sectional dimension
less than 1 mm, the particles being microparticles having a
cross-sectional dimension less than 100 microns. An apparatus may
further include a particle characterization device, operable to
characterize manipulated particles.
An example apparatus may be or further include a cytometer,
fluorescence particle detector, particle sorter, fluorescent
spectrometer, genetic analyzer, chromatograph,
electrophoresis-based detector, biomarker detector, blood
fractionator, or blood plasma fractionator. Example apparatus
include portable, point-of-care microfluidic diagnostic apparatus
for medical use. Blood separation can be used to assist diagnostics
of diseases through improved detection of clinical markers, such
detection of blood components such as protein components.
An example apparatus for manipulating particles within a fluid
sample, for example separating particles having different
characteristics, includes a substrate supporting a pair of
spaced-apart surface acoustic wave transducers configured to
generate a standing surface acoustic wave (SSAW) within a SSAW
substrate region located between the transducers, and a channel
configured to receive a fluid flow including particles, the channel
having a SSAW region where the channel has a SSAW channel region
where the channel passes proximate or adjacent the SSAW substrate
region. The substrate may form a wall of the channel, or the
channel may be bonded to the substrate within the SSAW substrate
region. The SSAW has an SSAW direction at an oblique angle to the
channel direction. In this context, the SSAW direction is a
direction parallel to linear nodes of the SSAWs. The apparatus is
operable to sort particles within the fluid sample when the fluid
sample is introduced into the channel and the SSAW is generated.
Pressure nodes and antinodes are generated perpendicular to a line
between the generators. Particles may be selectively directed to
nodes or antinodes, depending on particle properties. The physical
separation of the particle streams may be controlled through the
flow direction, angle of the SSAW to the channel, flow speed,
and/or other control parameters. The physical separation may be
matched to the separation of a pair of output channels, so that
particles directed to pressure nodes exit through one outlet
channel, and particles not directed to nodes or directed to
anti-nodes exit through the other outlet channel.
An example device includes a pair of interdigital transducers
(IDTs, also referred to as interdigitated transducers) supported by
a piezoelectric substrate. An IDT may comprise two interlocking
comb-shaped electrodes, the electrodes being provided by a metal or
other conducting coatings supported by the substrate. The
piezoelectric substrate may comprise a ferroelectric material such
as lithium niobate, and the IDTs may be deposited on a lithium
niobate substrate.
Particle suspensions (such as microparticle and/or nanoparticle
suspensions) are introduced through a channel located between two
IDTs. The channel may be formed in a polymer, such as PDMS. For
example, the channel may be formed by a molded polymer element on
the substrate, and may be a microchannel. The molded polymer
element may additionally include a cut-out (area in which it does
not contact the substrate) so as to define the SSAW region of the
substrate. A radio-frequency signal is applied to each IDT, which
then generates a SAW that propagates toward the channel. The
interference of the SAWs results in the formation of a SSAW on the
substrate.
An example particle manipulation apparatus comprises a substrate,
at least one surface acoustic wave (SAW) generator operable to
generate a standing surface acoustic wave (SSAW) in the substrate;
and a channel configured to receive a fluid sample including
particles, the channel having a particle manipulation region
located on a portion of the substrate in which the SSAW is
generated. Methods and apparatus according to embodiments of the
present invention may further include particle characterization,
for example using radiation directed at a manipulated particle
flow, or manipulated particles within a static fluid sample.
In another example, a particle manipulation apparatus or method in
accordance with the present invention utilizes other forms of
acoustic waves, such as bulk acoustic waves, wherein the waves are
at an oblique angle to the channel and/or flow direction. In any of
the embodiments discussed herein, other types of acoustic waves may
be substituted for the surface acoustic waves described.
Particle characterization may include apparatus and methods for
particle detection, particle analysis, particle counting, and
combinations of such approaches. For example, a radiation source
may be used to direct radiation towards manipulated particles
within a fluid medium. The integration of particle manipulation
with analytical methods and apparatus allows improved methods and
apparatus for particle characterization. Particles may be suspended
in the fluid medium, which may be a sample flow through the
channel.
For example, the integration of microfluidics devices with single
microparticle detection techniques allows improved microparticle
characterization. Examples of the present invention include
apparatus and methods for flow cytometry, and apparatus for
counting, analysis, and sorting of microparticles in a sample flow.
Microparticles may be defined as particles having a dimension of
less than 1 mm, in particular less than 500 microns, and more
particularly less than 100 microns. Microparticles may include
cells, molecules, biomolecules, and the like.
Examples of the present invention include improved flow cytometers
and other cell characterization devices, improved molecule
detection devices, other analyte characterization devices, analyte
sorting devices, genetic analysis devices, and the like. A SAW
(SSAW or propagating SAW) can be used for dynamic particle
separation and subsequent sorting. A particle may be a molecule
(such as a polymer, macromolecule, or biomolecule), biological
structure (such as a cell, for example a blood cell), particle (of
any type), micelle, droplet of different density from a host fluid,
and the like.
Apparatus and methods in accordance with the present invention may
be used for a wide variety of applications. The apparatus and
method may be used in almost all applications in which different
components have a difference in size or density or mechanical
properties. Some non-limiting examples include: separation of
different components (red blood cells, white blood cells,
platelets, plasma, etc) of a blood sample; separation of
circulating tumor cells from a blood sample; separation of
circulating endothelial cells from a blood sample; separation of
protein biomarker bound particles from a blood sample; separation
of microvesicles/exosomes bound particles from a blood sample;
separation of fetal nucleated erythrocytes from a maternal blood
sample (based on size and deformability); stem cell isolation based
on size differences; and bacteria enrichment from a blood sample.
Other applications will be clear to those of skill in the art.
An apparatus may be a planar microfluidic device. A channel may
have a lower wall parallel to and proximate the substrate, opposed
side walls, and an upper wall. A channel width and/or height may be
in the range 100 nm-1 mm, for example in the range 1 micron-500
microns. Other dimensions are possible.
A piezoelectric substrate may comprise lithium niobate, lithium
tantalate, lead zirconium titanate, polymer such as polyvinylidene
fluoride (PVdF) or other fluoropolymer, quartz, or other material.
An IDT can also form part of a sensor system, for example using
time gating or monitoring drive signal properties. In some
examples, the substrate may provide a wall of the flow channel, or
the flow channel may have a wall bonded to the substrate.
Patents, patent applications, or publications mentioned in this
specification are incorporated herein by reference to the same
extent as if each individual document was specifically and
individually indicated to be incorporated by reference. In
particular, the entire content of application Ser. No. 12/631,059,
filed Dec. 4, 2009, is incorporated herein by reference.
The invention is not restricted to the illustrative examples
described above. Examples are not intended as limitations on the
scope of the invention. Methods, apparatus, compositions, and the
like described herein are exemplary and not intended as limitations
on the scope of the invention. Changes therein and other uses will
occur to those skilled in the art. The scope of the invention is
defined by the scope of the claims, including all equivalents.
* * * * *